The aim of the present work was to determine the modulatory role of age and sex factors on the hypothalamo-pituitary-adrenal (HPA) axis in mice. For this purpose, HPA axis function was evaluated in 2- and 15-month-old mice of both sexes treated intraperitoneally with: (a) insulin (INS; 0.3 IU/mouse); (b) CRH (0.5 mu g/mouse); (c) ACTH (0.1 mu g/mouse) or (d) vehicle alone (50 mu l). Mice were then killed by decapitation either 20 min (ACTH), 30 min (CRH) or 45 min (INS) after treatment. Trunk blood was collected and adrenal glands were excised; plasma glucose (G), ACTH and corticosterone (B) levels as well as adrenal B content were determined by specific assays. The results indicate that, although no age-or sex-related differences in basal and stimulated ACTH secretion were found, adrenal glucocorticoid content and output were significantly higher in females than in males in both basal and post-stimuli conditions, regardless of the age of the animals. Stimuli-induced B production (content and secretion) was also found to be dimorphic with respect to sex. Adrenal B release after stimuli was significantly higher in 2- than in 15-month-old mice of both sexes. Although no age-dependent difference in stimuli-enhanced adrenal glucocorticoid content was observed in males, adrenal corticosterone content was higher in 15- than in 2-month-old female animals. Our results clearly indicate that: (a) females of different ages showed a higher HPA axis function than the respective groups of age-matched males and (b) 2-month-old animals showed higher HPA function than 15-month-old animals, regardless of sex. These results suggest that both age and sex factors play modulatory roles in HPA axis activity in both basal and post-stimuli conditions. In summary, our study strongly indicates a clear sexual dimorphism during adulthood and middle age, and suggests that the decrease in HPA axis function during middle age could be due to a failure of the adrenocortical cell function. These findings could be of relevance for steroid therapy applied in aging patients with certain pathophysiological conditions.